A Method to Determine Xenobiotic Acetylation Rate by Taq SNP rs1495741.

Drug acetylation plays an important role in the medical practice. Modern methods of acetylation phenotype prediction are based on genotyping of polymorphisms in the second exon of the gene NAT2. Some disadvantages of these methods limit their application in the clinical practice. We developed a method of human genotyping based on identification of NAT2 gene polymorphism rs1495741 by real-time PCR. This method of genotype determination has a number of advantages: high sensitivity, simplicity, possibility of automated interpretation of the results, and feasibility in clinical laboratories.

A Method for Evaluation of the Level of Circulating Mitochondrial DNA by ND1 and ND2 Genes.

The measurement of the level of mitochondrial DNA (mtDNA) in the blood is a difficult problem due to high variability of mitochondrial genes, deletions in the mitochondrial genome in some pathological conditions, different sources of mtDNA into the bloodstream (mtDNA from tissues, from blood cells, etc.). We designed primers and TaqMan probes for highly conserved regions of the ND1 and ND2 genes outside the mitochondrial deletions "hot zones". For standardizing the technique, the true concentration of low-molecular-weight mtDNA was determined by real-time PCR for two targets: a fragment of the ND2 gene (122 bp) and the ND1 and ND2 genes (1198 bp). The sensitivity and specificity of the developed approach were verified on a DNA pool isolated from the blood plasma of healthy donors of various nationalities. The concentration of low-molecular-weight mtDNA in the blood plasma of two patients with COVID-19 was monitored over two weeks of inpatient treatment. A significant increase in the content of low-molecular-weight mtDNA was observed during the first 5 days after hospitalization, followed by a drop to the level of healthy donors. The developed technique makes it possible to assess the blood level of low-molecular-weight mtDNA regardless of the quality of sampling and makes it possible to standardize this biological marker in a wide range of infectious and non-infectious pathologies.

Search for Effective Serum Tumor Markers for Early Diagnosis of Hepatocellular Carcinoma Associated with Hepatitis C.

The aim of the study was to identify the most effective serum tumor markers for early diagnosis of hepatocellular carcinoma based on the combination of diagnostic characteristics and correlations. Materials and Methods: There were observed 55 patients with chronic hepatitis C in the stage of liver cirrhosis with a verified diagnosis of hepatocellular carcinoma. The control group consisted of 55 patients with chronic hepatitis C at the stage of liver cirrhosis without hepatocellular carcinoma, comparable to the experimental group in terms of basic clinical profile. The following tumor markers were estimated in both groups: alpha-fetoprotein (AFP), alpha-fetoprotein-L3 (AFP-L3), annexin A2 (ANXA2), heparin-binding growth factor Midkine (MDK), glypican-3 (GPC3), des-gamma-carboxyprothrombin (DCP, PIVKA-II), dickkopf-related protein 1 (DKK-1), osteopontin (OPN), and Golgi protein 73 (GP73). There were also evaluated such indices as diagnostic sensitivity, specificity, positive predictive value, negative predictive value, likelihood ratio of a positive test, the possible correlation between alpha-fetoprotein and other tumor markers. The area under the ROC curve (AUC) was calculated at the 95% confidence interval. Results: The greatest sensitivity was revealed when using heparin-binding growth factor, annexin A2, osteopontin. Alpha-fetoprotein, alpha-fetoprotein-L3, glypican-3, des-gamma-carboxyprothrombin, dickkopf-related protein 1 had the best specificity. AUC>0.75 was found in annexin A2, heparin-binding growth factor, glypican-3, des-gamma-carboxyprothrombin, osteopontin, Golgi protein 73. The likelihood ratio of a positive test result was the highest for glypican-3. A significant correlation was found between alpha-fetoprotein and alpha-fetoprotein-L3, annexin A2, des-gamma-carboxyprothrombin. Conclusion: According to the aggregate indicators of diagnostic efficiency, heparin-binding growth factor, glypican-3, and osteopontin are the most promising tumor markers of those studied. When they are used, integral AUC values are above the average, the level of these tumor markers in the blood of patients with hepatocellular cancer does not correlate with alpha-fetoprotein. They are applicable for diagnosing liver cancer in AFP-negative patients. The combined use of AFP + GPC3, AFP + OPN has already shown their advantages. However, the efficacy of the combination of AFP + MDK, GPC3 + OPN has not been determined yet; therefore, significance of the combined use of these tumor markers in the diagnosis of liver cancer should be investigated in the near future.

Clinical and Experimental Evaluation of Diagnostic Significance of Alpha-Fetoprotein and Osteopontin at the Early Stage of Hepatocellular Cancer.

We evaluated the possibility of using an experimental model of hepatocellular carcinoma to study oncomarkers of primary liver cancer and compared the diagnostic efficacy of alpha-fetoprotein and osteopontin in the experiment and in clinical practice. Experimental studies were performed on a model of hepatocellular carcinoma induced by administration of diethyl nitrosamine to Fisher-344 rats. In addition, the levels of α-fetoprotein and osteopontin were determined in 35 patients with hepatocellular carcinoma detected at stages I-II according to TNM classification. The proposed model of liver cancer in rats reflects the sequence of stages characteristic of hepatocellular carcinoma in humans: liver fibrosis-cirrhosis-cancer. This model is applicable for the study of tumor markers at the early stage of tumor development. Osteopontin was found to have a more powerful diagnostic potential then alpha-fetoprotein.

[Assessment of external risk factors of hepatocellular cancer development and markers of genetic predisposition to its development in the ethnic group of yakut - men].

AIM: To establish the main external and genetically determined risk factors for the development of hepatocellular cancer in the ethnic group of male Yakuts living in the Republic of Sakha (Yakutia) [RS (Y)] in the epidemiologically unfavorable conditions of the incidence of viral hepatitis. MATERIALS AND METHODS: A total of 97 male Yakuts were examined, including 44 people diagnosed with hepatocellular cancer and 53 people diagnosed with chronic viral hepatitis. HCC risk factors were identified by analyzing medical records and questioning patients. In the experimental and control groups, genetic studies of single nucleotide polymorphisms of genes mapped on the X-chromosome and involved in the activation of antiviral immunity along the TLR7 signaling pathway were performed. RESULTS AND DISCUSSION: In 100% of patients with hepatocellular cancer, infection with hepatitis B, C, D viruses or co - infection with these agents was detected. Every fourth patient with HCC in the RS (Y) was infected with hepatitis D. The course of hepatocellular cancer associated with HDV was characterized by rapid progression of liver cirrhosis, development of portal hypertension, bleeding from varicose veins of the stomach and esophagus (36.4%) and edematous ascitic syndrome (63.6%). In addition to viral agents, additional risk factors for liver cancer were identified, such as alcohol abuse, overweight, diabetes mellitus, and smoking. Among the studied variation sites of genes localized on the X-chromosome and encoding the reaction of innate antiviral immunity, no genetic marker was found with a sufficient degree of confidence determining the likelihood of hepatocellular cancer developing. CONCLUSIONS: The high incidence of hepatocellular carcinoma of the male population in the RS (Y) is due to the widespread prevalence of parenteral viral hepatitis, especially viral hepatitis D. Due to the introduction of mass vaccination of the population against hepatitis B in the Russian Federation in the foreseeable future in the RS (Y) we should see a decrease in the proportion of hepatocellular cancer associated with hepatitis B and D viruses, and therefore the focus should be on the treatment and prevention of hepatitis C virus and non - infectious risk factors.

[Application of alpha-fetoprotein and osteopontin combination for early diagnosis of hepatocellular carcinoma associated with hepatitis C.]

Liver cirrhosis in the outcome of hepatitis C is the leading cause of hepatocellular carcinoma (HCC) in the world. Early diagnosis and timely treatment of HCC are important for reducing mortality and increasing life expectancy of patients with hepatocellular carcinoma. To assess the risk of HCC, the definition of alpha-fetoprotein (AFP) in the blood is most widely used, but low sensitivity limits its diagnostic value. In 2012, a new HCC biomarker - osteopontin (OPN), which is a secreted phosphoprotein that has a high affinity for integrins was proposed. The level of acute renal failure begins to rise in the early stages of malignancy, before the period of HCC detection by imaging methods, and has significantly better sensitivity than AFP. The purpose of this study is to evaluate the diagnostic efficacy of the combined determination of alpha-fetoprotein and osteopontin in prospective monitoring of patients with chronic hepatitis C in the advanced phase of liver fibrosis. Monitoring of 588 patients with hepatitis C was carried out from February 2013 to February 2019. HCC was detected in 55 of them (2.6% per year). The combination of 2 biomarkers showed better diagnostic efficacy than alpha-fetoprotein and osteopontin separately: AUC 0.85 (95% CI 0.80-0.90) versus AUC 0.63 (95% CI 0.57-0, 70) and AUC 0.82 (95% CI 0.77-0.88), respectively. This combination showed a sensitivity of 85.5% and made it possible to diagnose HCC with a prognostic level of a positive result of 72.3% at 19,4±0,8 weeks before the diagnosis was confirmed by instrumental imaging methods (ultrasound, MRI, CT). In the combined variant, ARF made the greatest contribution to the increase in diagnostic efficacy (AUC). At an early and very early stage of HCC development, isolated HCC elevations were found in only 5.4% of patients. Conclusion: the combined use of alphafetoprotein and osteopontin as a diagnostic panel can be recommended for monitoring patients with liver cirrhosis in the outcome of hepatitis C and predicting HCC at an early stage of development.

Population Polymorphism of IFNL3 and IFNL4 Genes of Type 3 Interferon Associated with Spontaneous Clearance of Hepatitis C Virus in Representatives of Caucasian and Mongoloid Races.

The frequency of single nucleotide polymorphisms of IFNL3 gene (rsl12979860 and rs8099917) and dinucleotide polymorphism of IFNL4 gene (ss469415590) were estimated in healthy inhabitants of Mongolia and Irkutsk regions taking into account their races. Population and genetic studies were performed in 1520 conventionally healthy volunteers (blood donors), representatives of Caucasian and Mongoloid races. Significant race-related differences in the incidence of IFNL3 and IFNL4 gene polymorphisms associated with spontaneous clearance of hepatitis C virus were found in healthy volunteers.

[EPIDEMIOLOGIC MANIFESTATIONS OF VIRAL HEPATITIS C IN REPRESENTATIVES OF CAUCASIAN AND MONGOLOID RACES].

AIM: Present comparative epidemiologic characteristics of viral hepatitis C in Mongolia and Irkutsk Region taking into account racial origin of the studied populations. MATERIALS AND METHODS: The studies were carried out in 2009-014 on the territory of Irkutsk Region in Mongolia. Prevalence of viral hepatitis based on serological monitoring, virus RNA detection, risk factors, change in structure of circulating genotypes, hepatocellular carcinoma morbidity were studied. RESULTS: Epidemiologic manifestations of viral hepatitis C in Mongolia, in contrast to Irkutsk Region, are characterized by, a wider prevalence of the disease, predominance of the fraction of seropositive individuals in age category of above 50 years and predominance of genotype 1 virus in circulation. In recent years an evolution of diversity of circulating' irus genotypes, ook place towards a reduction of the fraction of genotype in Mongolia and Russia due tor ni ncrease of the fraction of genotype-3. Expressed,differences in average-annual values of hepatocellular carcinoma morbidity were detected, that were more than 10 times higher among Mongoloids compared with Caucasians. CONCLUSION: Pronounced differences were detected in manifestations of epidemic process of viral hepatitis C in Mongolia and Asian part of Russia, represented by Eastern Siberia, that are associated with ethnic, social and, cultural living conditions of the indigenous population.

[Sanitary-Epidemiological Status of Siberian Population (Medico-Demographical and Epidemiological Characteristics)].

Review on the problem of sanitary-epidemiological welfare of the population in the Siberian Federal District (SFD) was conducted based on literature data and authors own research in the period of 2002−2014. Authors provided broad information on the health and demographic and epidemiological characteristics of SFD population. SFD in comparison with other regions of the Russian Federation overcomes one of the most adverse situations including mortality rates from external causes. SFD population's infectious and somatic morbidity rates were analyzed. Analysis demonstrated that the situation relating to priority epidemiologically and socially important infections (HIV-infection, parenteral viral hepatitis, tuberculosis etc.) on the territory of the SFD remains tense. Authors provided information on the increase in the level of the actual for Siberian regions natural-foci tick-borne infections. Detailed analysis for the environment anthropogenic pollution impact for the epidemic, infectious and vaccine induced processes. Authors suggest that anthropogenic (biological) environmental pollution is one of the most important factors influencing the epidemiological welfare of the Siberian population. A new strategic direction in epidemiological research associated with the problem of comorbid diseases is planned.

[The early diagnosis of neurodegenerative changes in multiple sclerosis and clinically isolated syndrome based on optical coherence tomography of the retina]. / Ranniaia diagnostika neirodegenerativnykh izmenenii pri rasseiannom skleroze na osnove programmy diagnostiki setchatki.

AIM: To study signs of neurodegeneration in the retina in multiple sclerosis. MATERIAL AND METHODS: We studied the retinal nerve fiber layer (RNFL) in 265 (524 eyes) patients. RESULTS AND CONCLUSION: We identified a reliable thinning of RNFL of both eyes in patients with relapsing-remitting multiple sclerosis (MS). A comparative analysis by sectors revealed a specific decrease in RNFL in temporal segments and papillomacular beam. Mean values of RNFL in patients with clinically isolated syndrome did not differ from those in controls. Thickening of RNFL of low segments (NI и TI) and decrease in the thickness of the upper temporal segment (TS) were found. The results have demonstrated that neurodegenerative signs in the retina may be early appearances of MS. Optical coherence tomography allows to perform objective and rapid screening of retinal neurodegenerative changes.

[Results of using biocoating by means of the amniotic sac. Communication 1. Keratoplasty with graft biocoating].

To protect a corneal graft at keratoplasty from tear moisture hyperhydration of the de-epithelized surface and from the negative exposure to contact with the eyelid mucosa in order to enhance surgical efficiency, the authors have developed a few biocoating technologies that involve amnionic sac placement on the graft, with its fixation on the eye of a recipient, by applying a full-thickness and layered corneal disc autologous flap or a ring-shaped allogenic corneoscleral one. Fifty-two patients (55 eyes) with primary (n=5) and secondary (n=33) corneal dystrophy and leukomas (n=14) were operated on. Penetrating keratoplasty and layer-by-layer grafting were performed in 38 (40 eyes) and 14 (15 eyes) patients, respectively. The biocoat was removed from the graft following 5-7 days; by that time complete epithelization of its anterior surface occurred in the majority of patients. After penetrating keratoplasty and layer-by-layer grafting, there was a transparent engraftment in 92.5 and 93.3% of patients, respectively. Visual acuity up to 0.2-0.6 restored in 74.5% of patients.

[Current aspects of the clinical picture of tick-borne encephalitis].

Based on the long-term follow-up of 655 patients with different forms of tick-borne encephalitis (TBE), the authors describe its present clinical characteristics in the Baikal natural focus. They have established the preponderance of the meningeal form of the infection and the relatively severe course with residual phenomena and a fatal outcome in patients with the poliomyelitic form of TBE.

[Glutathione system in erythrocytes and plasma in viral hepatitis].

In all 5 acute (AVHs) and chronic viral hepatites (CVHs) there was the increase of erythrocyte activities of glutathione peroxidase (GPx) and glutathione reductase (GR), and the decrerase in GSH concentration. In blood plasma there was accumulation of GPx, glutathione S-transferase (GST) and y-glutamyl transferase (gammaGT). GSH and GR increased in plasma only in AVHs. In CVH C erythrocyte GST increased. Evidently changes in the erythrocyte glutathione system are reactions to oxidative stress and in blood plasma they are consequences of inflammation and hepatocyte cytolysis. Changes were more pronounced in middle-heavy course than in the heavy one. These changes have pathogenic importance and can be used in addition to complex diagnostics. They are significantly differed from changes in chronic gall-bladder diseases. Necessity of separate investigation of glutathione system in erythrocytes and blood plasma but not in whole blood is argued.

[Impact of Yersinia pseudotuberculosis on the in vitro production of cytokines by whole blood cells of donors]. / Vliianie Yersinia pseudotuberculosis na produktsiiu tsitokinov kletkami tsel'noi krovi donorov in vitro.

The impact of two plasmid (47, 82 MD), single plasmid (47 MD) and non plasmid Y. pseudotuberculosis strains, Y. enterocolitica (47 MD) as well as Y. pseudotuberculosis superantigen (YPM) on the production of interleukin-1 (IL-1), interleukin-6 (IL-6), interferon-alpha (IFN = alpha) and tumor necrosis factor alpha (TNF-alpha) by whole blood cells obtained from donors was studied. All Y. pseudotuberculosis and Y. enterocolitica strains stimulated the production of IFN-alpha, IL-1, IL-6 and TNF-alpha by whole blood cells, but considerably less than Y. pseudotuberculosis lipopolysaccharide and YPM. These data are indicative of the pathogenetic role played by 82 MD plasmid in manifestation of Y. pseudotuberculosis immunosuppressive properties. The maximum stimulation of the production of cytokines was observed under the action of YPM, which confirmed an important role played by this superantigen in the pathogenesis of Y. pseudotuberculosis.

[An analysis of the antibodies to the outer cell membrane proteins in Yersinia in the varying course of human pseudotuberculosis]. / Analiz antitel k belkam naruzhnoi kletochnoi membrany iersinii pri razlichnom techenii psevdotuberkuleza u liudei.

Time course changes in the formation and spectrum of the antibodies to the proteins of Yersinia pseudotuberculosis external cellular membrane were studied by immunoblotting. A total of 154 samples of blood serum from 46 pseudotuberculosis (PT) patients were tested. 53 samples were obtained from PT patients with acute disease, 50 from PT patients with recurrences and 51 samples cam from PT subjects with protracted disease. Electrophoresis in polyacrylamide gel with sodium dodecyl sulfate disclosed a number of basic protein components with molecular characteristics: 22-26-33-37-45-59-67 kD. Irrespective of the disease clinical presentation, the antibodies were directed primarily to the protein component of 26 kD molecular mass this evidencing its immunodominant role. There appeared some specific features in formation rate and circulation time of the above antibodies. This finding may be utilized for diagnostic and prognostic purpose.

[Clinico-immunologic characteristics of complicated and uncomplicated yersiniosis]. / Kliniko-immunologicheskaia kharakteristika oslozhennogo i neoslozhnennogo iersinioza.

Asymmetric affection of the major lower limb joints is a characteristic feature of the joint syndrome in yersiniosis-associated arthritis. The sacroiliac articulations are frequently (47% cases) involved. In addition, yersiniosis-associated arthritis concurs with the signs and symptoms of systemic disease--gastroenterocolitis, myocardiopathy and myocarditis, erythema nodosum, hepatitis, urethritis, conjunctivitis, myositis and myalgia, enteropathy; changes in the CNS typical for the astheno-neurotic syndrome are frequently present. Comparison of the immunological assay data in complicated and uncomplicated yersiniosis shows equally high levels of IgG and CIC. High anti-DNA antibody titres are more frequently found in the serum of uncomplicated yersiniosis patients. ELISA quantitation of specific IgA, IgM, and IgG class antibodies in yersiniosis-associated arthritis patients demonstrated persistence of all the three antibody classes or of IgA-IgG combination in cases with most severe of the joint syndrome. In the presence of cardiac disease, patients were found to have high titres of antibodies reactive with the cardiac interstitial tissue, while in authentically diagnosed myocarditis cases with the sarcolemma. The investigation findings strongly suggest a high degree of involvement of immune and autoimmune processes in the pathogenesis of arthritides secondary to Yersinia infection.

[Autoimmune processes during the protracted course of human yersiniosis]. / Autoimmunnye protsessy pri zatiazhnom techenii iersinioza u liudei.

Altogether 187 blood sera from 87 patients with acute yersiniosis, 149 from 50 patients with a lingering course of yersiniosis, and 40 sera from healthy persons were investigated for the presence of antibodies to cells of interstitial myocardial tissue, fibroblasts of human embryonic articular fibrous tissue, nuclei and fibroblasts of human skin in pure culture by indirect immunofluorescence. Antibodies to the above structures were more frequently found in the blood sera of the patients with a lingering course of yersiniosis (p less than 0.001). In most cases antibodies belonged to IgG or IgG and IgM. Periods of exacerbation of an inflammatory process were combined with increased titers of antibodies to tissue components suggesting the pathogenetic importance of antibodies in the formation of a lingering course of yersiniosis.